The present disclosure relates generally to semipermeable membranes, in particular very thin membranes with nonrandom pores, and more particularly to tunable polymer nanofilm perfusion membranes, which are believed to be advantageous for use in microfluidic devices for cell biology studies.
The basal lamina or basement membrane is a key physiological system that supports diverse epithelial cell types and participates in physicochemical signaling and transport between tissue types [4]. Its formation and function are essential in tissue maintenance, growth, angiogenesis, disease progression, and immunology.
In vitro models of the basement membrane, such as Boyden and transwell chambers, are essential to cell biology studies and are common in lab-on-a-chip devices where cells require apical and basolateral polarization. Extravasation, intravasation, membrane transport of chemokines, cytokines, chemotaxis of cells, and other key functions are routinely studied using these models. For example, transwells have been widely adopted for perfused, polarized cells, and migration assays since Boyden's initial chemotaxis experiment [1].
The promise of organs-on-a-chip technology (i.e., 3-D microfluidic cell culture chips that simulate the activities, mechanics, and physiological response of entire organs and/or organ systems) is essentially to create a more robust in vitro model of the complex electrophysicochemical systems that control cell function and fate in multicellular organisms [2]. However, a key element of any such model, including both static transwells and complex microfluidic organ models, is the semipermeable membrane used to simulate the basement membrane [3].
Critical properties of a semipermeable membrane suitable for use in a microfluidic device include controlled porosity, high species flux, mechanical strength, surface biocompatibility, and optical transparency. Although semi-permeable membranes formed from polycarbonate (PC) and polyester (PE) have traditionally been used for transwell cell culture and diffusion experiments, such membranes are far thicker than the basement membrane. Transwell membranes are typically more than 100 microns thick and the basement membrane is much less than one micron thick, typically tens of nanometers, three to four orders of magnitude thinner than currently available polycarbonate and polyester membranes, the transparency of which is not optimal in bright field light and confounds observation by differential interference contrast.
By contrast, ultrathin polymer films having a thickness of less than one micron (referred to herein as “nanofilms”) are promising candidates for use as an in vitro model of the basement membrane because they are closer in thickness to native basement membrane and are compatible with cell culture. Nanofilms with thicknesses ranging from tens to hundreds of nanometers belong to a class of polymeric nanomaterials having huge surface to thickness ratios (>106), unique thickness-dependent interfacial and mechanical properties, and optical transparency. Previous studies have revealed that such films can have non-covalent high adhesiveness to surfaces, tunable flexibility and molecular permeability [5], defined structural color [6] and mechanical strength [7], and possibly conductive [8] and magnetic [9] properties, all of which enable such nanofilms to closely mimic the lamina basalis of the extracellular matrix in human tissues, making such films an ideal structure to direct cellular organization and regulate organ regeneration and function in vitro.
Nanofilm fabrication techniques are diverse, and include layer-by-layer assembly [10] methods, and the Langmuir-Blodgett method [11], among others. However, the simplest route to fabricate freestanding and easy-to-handle nanofilms is spin coating from a liquid polymer-solvent solution [12]. In previous works, full characterization of plain nanofilm structures was performed. The adhesion and proliferation of different cell types on poly (lactic acid) (“PLLA”) nanofilms [13] and polyelectrolytic films [14] was confirmed.
Existing examples of organs-on-a-chip have used perforated polydimethylsiloxane (“PDMS”) membranes [15, 16], polycarbonate [17] or photoresist membranes [18]. While the PDMS membranes have the advantages of optical transparency, tunable elastic modulus, and can be easily integrated in a monolithic PDMS device, it is still difficult to reduce the thickness of the membrane below 5 μm. This represents a major drawback of PDMS membranes because submicron membrane thickness is essential for physical contact and paracrine communication between cells growing on both sides.
Numerous approaches have been used to fabricate organic semipermeable membranes [19], such as polymer synthesis [20] and ion track etching of polymers [21]. However, these methods have numerous drawbacks mainly related to random arrangement of pores with wide size distribution or nanometric pore size. Other methods are based on direct micro- and nanofabrication on the plain polymer sheet, by e-beam lithography and by focused ion beam milling [22, 23]. These approaches have the advantage of control of the design and arrangement of the pores [24], but they also present drawbacks, such as long working time and seriality, and thus high cost.
Moreover, while direct etching of any polymeric film should be theoretically possible for example by tuning current and voltage of an ion or electron beam [24], it is practically impossible to drill holes with micrometric diameter through an approximately 100 nm thin film because the Gaussian distribution of the ion beam and the control of current and milling depth are not sufficient to avoid local polymer melting. Debris from the milling process limits the depth to 10× the diameter of the hole. Furthermore, direct milling of the film would implicate local changes in the film's mechanical and structural characteristics and thus increase the risk of breakage and tears in the polymer film.
Accordingly, what is needed are improvements in semipermeable membranes for use in microfluidic devices.